Glycoproteins, glycolipids and polysaccharides are present on the cell surface of mammalian cells and are central molecules in many biological processes. They participate in cell-cell recognition, cell differentiation and various receptor-ligand interactions throughout biology. Many of these biologically active glycans contain an essential 9-carbon sugar that is known as sialic acid, or N-acetyl-neuraminic acid (NeuAc).
Some bacterial pathogens that invade the mammalian host have taken advantage of the presence of sialic acid containing glycoconjugates on the host. These bacteria display some of these same carbohydrate chains on bacterial cell surfaces, and indeed a role for these carbohydrates in pathogenesis has been demonstrated. See, e.g., Kahler, C. M. and Stephens, D. S., Crit. Rev Microbiol, 24:281-334 (1998), and Moran, A. P. et al., FEMS Immunol Med Microbiol, 16:105-115 (1996). It is thought that the presence of the carbohydrate mimics allows the pathogens to escape detection by the immune system since these molecules are not considered foreign. Further, the presence of these carbohydrates presents a physical barrier for the killing action of serum complement. See, e.g., Vogel, U. et al., Med Microbiol Immunol (Berl), 185:81-87 (1996). Finally it may be that certain pathogens use normal human receptors that recognize their surface carbohydrate structures as a means of aiding transmission (or colonization of the host, although this mechanism remains unproven for many of these pathogens). See, e.g., Preston, A. et al., Crit Rev Microbiol, 22:139-180 (1996) and Harvey, H. A. et al., Mol Microbiol, 36:1059-1070 (2000).
Capsular polysaccharides from group B Neisseria meningitidis and Escherichia coli K1 have sialic acid in linkages that are molecular mimics of the polysialic acid (PSA) structure seen mainly in the mammalian neural cell adhesion molecule, a brain specific protein integral to neuronal function. Thus they are found as a homo-polymer of α-2,8-linked Neu5Ac, and also as homo-polymers of α-2,9-linked residues, as a co-polymer in which the linkage is mixed α-2,8/α-2,9, and finally as polymers in which other sugars are included, as in the Group Y and W Neisseria meningitidis. These polysialic acid capsules are required for neuro-invasive disease in the case of E. coli, N. meningitidis and P. haemolytica. See, e.g., Silver, R. P., and E. R. Vimr. 1990. Polysialic acid capsule of Escherichia coli K1, p. 39-60. In B. H. Iglewski, and V. L. Clark (ed.), Molecular basis of microbial pathogenesis. Academic Press, Inc., San Diego, Calif. It is important to note that because many of these pathogens are specific for a human host, data from animal model infections may not have shown all of the true functions of these glycoconjugates.
To date there has been little detailed work on the fundamental aspects of the sialyltransferase enzymology from bacterial pathogens. It is possible to express, purify and crystallize some of those enzymes responsible for LOS sialylation. See, e.g., Gilbert, M. et al., J Biol Chem, 271:28271-28276 (1996); Gilbert, M. et al., J Biol Chem, 275:3896-3906 (2000); Chiu, C. P. et al., Nat. Struct. Mol. Biol., 11:163-170 (2004); and Yu, H. et al., J. Am. Chem. Soc., 127:17618-17619 (2005). However no such work has been done with those enzymes involved in the generation of the sialic acid homopolymeric capsules.
The genetic loci for the PSA capsule production have been identified in both E. coli and N. meningitidis, and some work has been done on the recombinant enzymes (NeuS) from E. coli K1, and K92. See, e.g., Cho, J. and Troy F A, I. I., PNAS, 91:11427-11431 (1994) and Shen, G. J. et al., J. Biol. Chem., 274:35139-35146 (1999). But again no detailed enzymology on the isolated sialyltransferase has been reported. The study of the enzymology has been hampered by the poor solubility of the enzyme thus hampering the production of polysialic acid conjugates in vitro. The present invention solves this and other needs.